Resolution of amino acids

ABSTRACT

A process for resolution of a mixture of D- and L- amino acids, selected from threonine (THR), asparagine (ASN), p-hydroxyphenylglycine p-toluene sulfonate (pHPGpTS) and glutamic acid hydrochloride (GLU), which crystallize in the form of a conglomerate, whereby the ratio of one desired enantiomorph to the other undesired enantiomorph of said amino acid is increased in the crystalline compound obtained, as compared to the ratio in the starting material, which process comprises forming a supersaturated solution of said mixture, adding another predetermined amino acid as additive, which has a molecular structure which resembles that of one of the enantiomers of said racemic mixture, said additive being a D-amino acid as an inhibitor of the growing D-amino acid when the L-amino acid is desired, or a L-amino acid when the D-amino acid is desired, and crystallizing part of the compound from said supersaturated solution. When GLU.HCl is resolved, the crystals can be separated as they have different morphological forms.

The present invention relates to a novel process for the resolution ofamino acids from mixtures of the D- and L- forms, by crystallizationfrom supersaturated solutions, in the presence of an additive, whichresults in the preferred crystallization of a predetermined desiredform, in preference to the other. This can be considered as applicationof the kinetic resolution of racemates crystallizing in the form ofconglomerates by carrying out the crystallization in the presence ofsmall amounts of resolved additives, the stereochemical molecularstructure of which resembles that of one of the enantiomers of the saidracemic mixture.

This invention relates to the application of a similar principle to theresolution of four amino acids: threonine (THR), asparagine (whichcrystallizes from water as its monohydrate) (ASN),p-hydroxyphenylglycine-p-toluene sulfonate (pHPGpTS) and glutamic acidas its hydrochloride (GLU.HCl). Racemic mixtures of all four crystallizein the form of conglomerates.

It is known that all of the above compounds can be resolved by bringingaqueous solutions of their racemic mixtures to conditions ofsupersaturation, and then inducing preferential crystallization of thedesired enantiomer by introducing seed crystals of this enantiomer. Theinvention described herein is based on a different principle.

We have found that addition of D-glutamic acid (D-GLU), D-asparagine(D-ASN), D-aspartic acid (D-ASP), or D-cystein (D-CYS), or D-glutamine(D-GLN) in small amounts to a supersaturated solution of threonine inwater brings about a preferred crystallization of the threonine in itsL-form. Similarly, inclusion of the L-forms of the above additives leadsto preferred crystallization of D-threonine. Further, we have found thatfrom a supersaturated aqueous solution of D,L-asparagine, on addition ofD-aspartic acid, D-glutamic acid or D-glutamine, there results apreferred crystallization of L-asparagine monohydrate; inclusion of theL-additives leads to preferred crystallization of D-asparaginemonohydrate. Further, we have found that addition of L-tyrosine (TYR),L-tyrosine-p-toluene sulfonate (TpTS), L-Dopa, L-Dopa-p-toluenesulfonate (DpTS), L-α-methyl Dopa, L-α-methyl Dopa-p-toluene sulfonate(MDpTS), L-phenylalanine (PHE), L-phenylalanine-p-toluene sulfonate(PpTS), L-phenyl glycine (PG), L-phenyl glycine p-toluene sulfonate(PGpTS), L-p-methoxy phenyl glycine (pMPG) or L-p-methoxy phenyl glycinep-toluene sulfonate (pMPGpTS) to a supersaturated solution of pHPGpTS inan 0.5 N aqueous solution of p-toluene sulfonic acid causes preferredprecipitation of the D-form of pHPGpTS. On acidification thecorresponding enantiomer of p-hydroxyphenylglycine can be obtained.Inclusion of the D-additives causes preferred precipitation of theL-form of pHPGpTS.

We have found that addition of (D) lysine HCl or (D) lysine [(D) LYS],(D) ornithine HCl or (D) ornithine [(D) ORN], (D) histidine HCl or (D)Histidine [(D) HIS] in small amounts to a supersaturated solution of(D,L) GLU in 5N HCl, brings about preferential crystallization of GLU inits (L) form in high enantiomerical purity. Furthermore the addition of(D) Serine [(D)-SER], (D) Threonine [(D)THR], (D) Cysteine:HCl [(D) CYSHCl] in amounts larger than 20% of the amount of (D,L)GLU to thesupersaturated solution, brings about as well preferentialcrystallization of (L) GLU.

Similarly, addition of the (L) forms of the above additives leads topreferred crystallization of (D) GLU.HCl.

In all the above cases, relating to glutamic acid, the enantiomercrystallizing first (of absolute configuration opposite to that of theadditive), appears in the form of big well shaped bars and iscontaminated by amounts lower than 0.1% of the additive, which can beremoved by washing the crystal with solvent. The antipode can beobtained in the form of thin plates or powder by further crystallizationof the residual mother liquor, and contains about 0.5% of the additive.

Alternatively, from extensive crystallization of the supersaturatedsolution the two morphological forms, namely bars and plates, can beobtained together and then mechanically separated, leading to resolutionof the enantiomers of GLU:HCl.

In all the above cases crystals enriched in the second enantiomer can beobtained by crystallization from the residual mother-liquor, afterseparation of the first batch of crystals of the enantiomericallyenriched product.

DETAILED DESCRIPTION

This invention can be used to produce crystalline threonine, asparaginemonohydrate, pHPGpTS and glutamic acid, enriched in the desiredenantiomer, without requiring the use of seed crystals of thisenantiomer. The use of seed crystals from this enantiomer or from theracemic mixture may, however, be desirable from the point of view of therate of crystallization.

For the case where a seed crystal of the desired enantiomer is used,this invention describes an improvement of the resolution process forthreonine, asparagine, pHPGpTS, and glutamic acid HCl by furtheraddition in solution of the appropriate additives for each amino acid asmentioned above.

P The examples which follow are illustrative of the present inventionbut are not to be interpreted in a limiting sense.

EXAMPLE 1 Threonine

300 mg DL-threonine and 30 mg D-glutamic acid were slurried in 1 cc H₂O, and heated to about 80° C. until complete dissolution occurred. Thehot solution was filtered and cooled to room temperature withoutagitation. After 40 hrs 53 mg of crystals had formed, and these wereseparated by filtration. The specific rotation of these product crystalswas [α]_(D) ²⁰ =-25.6 (c5, H₂ O), corresponding to an enantiomericexcess (e.e.) of 91% L-threonine in the crystals in a yield of 18% withrespect to the initial D,L-threonine introduced. The crystals containless than 1% D-GLU (high performance liquid chromatography). A secondfiltration of the mother liquors, after three days, gave 50 mg ofcrystals having [α]_(D) ²⁰ =+25.4 (c5, H₂ O), corresponding to 91% e.e.of D-THR in a yield of 17%.

EXAMPLE 2 Threonine

The solution of D,L-threonine and D-glutamic acid, prepared as inExample 1, was thermostated at 67° C. for 30 min, after which powderedcrystals of L-THR (15 mg, 5%) were added and the resultant slurry wastransferred to a thermostat at 25° C. where it was shaken for 60 min.Crystals (59 mg) separated and were filtered off; they had [α]_(D) ²⁰=-27.1 (c2, H₂ O), corresponding to an e.e. of 94.8% of L-THR in theprecipitated material (crystals additional to the seeds), in a yield of14.6%.

EXAMPLES 3-8 Threonine

In a manner similar to examples 1 and 2, experiments were performed alsoat room temperature with 300 mg, D,L-THR/c.c. H₂ O, and with otheradditives. The conditions and results are given in Table I below. Inexamples 7 and 8 the crystallizations were performed with shaking, inexamples 3-6 without.

                  TABLE I                                                         ______________________________________                                        Resolution of threonine in the presence of various additives                           Weight            Precipitated Product                               Exam-          %        Seed  Time e.e. THR                                                                             Yield                               ple   Additive Additive*                                                                              L-THR (hr) (%)    (%)                                 ______________________________________                                        3     D-ASN    10       None  40   90   L   16                                4     D-ASP    10       none  40   70   L   16                                5     L-GLN    10       none  40   90   D   15                                6     L-CYS    13       none  40   93   D   7                                 7     None     --       5%     1   5.5  L   29.7                              8     D-GLU    10       5%     2   94.4 L   14.8                              ______________________________________                                         *relative to D,LTHR.                                                     

EXAMPLE 9 Asparagine

300 mg D,L-asparagine.H₂ O and 20 mg D-asparatic acid were slurried in 3cc H₂ O and the slurry heated to about 80° C. until complete dissolutionoccurred. The hot solution was filtered and cooled to room temperaturewithout agitation. After 100 hr the separated crystals (49 mg) wererecovered by filtration, and had [α]_(D) ²⁰ =+24 (c4, HCl 5N)corresponding to an e.e. of 79% of L-ASN in the crystals in a yield of16%. The filtered crystals contained 1.2% D-ASP.

EXAMPLE 10 Asparagine

300 mg D,L-ASN H₂ O and 50 mg L-ASP were processed as in example 9. Tothe filtered, cool solution (supersaturated) was added 0.5 mg ofcrystalline, powdered D,L-ASN.H₂ O

After 20 hr 42 mg of crystals were recovered by filtration, and had[α]_(D) ²⁰ =-29.9 (c4, HCl 5N) corresponding to an e.e. of 98% of D-ASNin the crystals in a yield of 14%.

EXAMPLES 11-14 Asparagine

In a manner similar to example 9, experiments were performed, also atroom temperature and with 100 mg D,L-ASN.H₂ O/cc water, with otherimpurities. The conditions and results are summarized in Table II.

                  TABLE II                                                        ______________________________________                                        Resolution of asparagine in the presence of various additives                           Weight %                                                                              Time   Precipitated Product                                 Example                                                                              Additive Additive* (hr) e.e. ASN (%)                                                                           Yield %                               ______________________________________                                        11     L-GLU    15        90   85 D     22                                    12     L-GLN    15        17   10 D     35                                    13     D-GLU    15        90   79 L     14                                    14     none     15        48   0        42                                    ______________________________________                                         *relative to D,LASN.H.sub.2 O.                                           

EXAMPLE 15 pHPGpTS

300 mg D,L-pHPGpTS and 22 mg L-tyrosine were slurried in 1 cc 0.5Mp-toluenesulfonic acid in water, and the slurry was heated untilcomplete solution occurred. The hot solution was filtered and allowed tocool to room temperature without agitation. After 5 hr spontaneouscrystallization started. After an additional 1.5 hr, 57 mg of crystalsof pHPGpTS were collected by filtration; they had [α]_(D) ²⁰ =-50.5 (c2,H₂ O) corresponding to an e.e. of 75% of D-pHPGpTS in 19% yield. Thecrystals contained only traces of L-TYR.

EXAMPLE 16 pHPGpTS

The initial solution was made up as in example 15, and seeded at 30°with 1.5 mg (D-pHPGpTS (0.5%). The slurry was held at 30° C. in athermostat, with gentle shaking. After 1 hr, 17.27 mg of crystals werecollected by filtration, with [α]_(D) ²⁰ =-66.5, corresponding to ane.e. of 98.7% of D-pHPHGpTS in 5.3% yield.

EXAMPLES 17-21 pHPGpTS

In a manner similar to example 16, experiments were performed withdifferent additives and conditions. The results are summarized in TableIII below. In all cases the amount of seeds of D- or D,L- pHPGpTS was0.5 weight % of the initial D,L-pHPGpTS. The additive was always 15weight % of the initial D,L-pHPG.

                                      TABLE III                                   __________________________________________________________________________    Resolution of pHPGpTS in the presence of various additives                    [D,L-                           Purified Product                              pHPGpTS]                    Time                                                                              .THorizBrace.                                 Example                                                                            (mg/cc)                                                                             Additive                                                                             Conditions*                                                                             (min.)                                                                            c.c. (%)                                                                           Yield (%)                                __________________________________________________________________________    17   350   L-TYR  D-seeds at 50° C.                                                                120 61.4 24.5                                                       followed by                                                                   shaking at 30° C.                                    18   350   L-TYR  D-seeds at 50° C.                                                                60  100  5.3                                                        followed by                                                                   shaking at 25° C.                                    19   300   L-DOPA D,L-seeds at 30° C.                                                              90  19.7 31.8                                                       standing at 20° C.                                   20   300   L-PHE  D,L-seeds at 30° C.                                                              90  31.3 20.8                                                       standing at 20° C.                                   21   300   L-MeDOPA                                                                             D,L-seeds at 30° C.                                                              90  19.3 33                                                         standing at 20° C.                                   __________________________________________________________________________     *D-seeds obtained by recrystallization of the Dsalt from MeOH.           

EXAMPLE 22 Resolution of (D,L) pHPGpTS by addition of Phenyl Glycine(PG)

350 mg (D,L) pHPTpTS and 24 mg Phenyl Glycine (PG) were slurried in 1 cc0.5 M p-toluene sulphonic acid and treated as in example 15. After 2hours the supersaturated solution was seeded with 1 mg (L) pHPGpTS.After 1 additional hour, the resulting crystals were collected byfiltration. The enantiomeric excess was 80.2% and the yield 19.4%.

Addition examples of resolution of pHPGpTS by addition of PG or pMPG in15 weight % of the initial (D,L) pHPG are given in Table IV below:

                  TABLE IV                                                        ______________________________________                                        (D,L)                         Purified                                        pHPGpTS                       Product                                         Example                                                                              (mg/cc)   Additive  Conditions                                                                             e.e. Yield                                ______________________________________                                        23     350       (D)PG     seeds, (D,L)                                                                           78   17.5                                                            room temp.                                         24     350       (D)pMPG   seeds (D,L)                                                                            69   12.3                                                            room temp.                                         25     350       (D)pMPG   seeds (L)                                                                              80   16.3                                                            room temp.                                         ______________________________________                                    

EXAMPLE 26

1 g (D,L) GLU.H₂ O and 200 mg (L) LYS HCl were slurried in 5 ml 5N HCland heated to about 60° C. until complete dissolution occurred. Thesolution was filtered, cooled to room temperature, and about 0.5 mg seedcrystals of (D,L) GLU.HCl added. After one day of standing withoutagitation, 229 mg crystals had formed, and these were separated bydecantation and dried. The specific rotation of these product crystalswas [α]_(D) ²⁵ =-24.2° (c=5, HCl 1N), corresponding to an enantiomericexcess of 98.4% (D) GLU in an overall yield of 20.7% with respect to theinitial (D,L) GLU introduced.

EXAMPLES 27-30

In a manner similar to Example 26, experiments were performed also atroom temperature in 5N HCl in the presence of (L) LYS.HCl and seeds of(D,L) GLU.HCl but in different conditions. The results are given inTable V below:

                  TABLE V                                                         ______________________________________                                        Resolution of (D,L) GLU.HCl in the presence of (L) LYS HCl                                                       Precipitated                               Ex-                 wt.            crystals                                   am-  wt. of (D,L)GLU.HCl                                                                          LYS.HCl  Time        overall                              ple  in 5 ml 5N HCl (g)                                                                           (mg)     (days)                                                                              e.e.  yield                                ______________________________________                                        27   1               25      1     96.3  14.8                                 28   1              200      1     90.2  22.6                                 29   1.6            333      4     100   20.8                                 30   1              800      10    99.1  20.2                                 ______________________________________                                    

EXAMPLE 31

1 g. (D,L) GLU.H₂ O and 100 mg (L) ornithine HCl were slurried in 5 ccHCl 5N, and the slurry heated until complete dissolution occurred. Thesupersaturated solution was filtered, cooled to room temperature, about0.5 mg (D,L) GLU.HCl crystals were added.

After standing of the solution 2 days without agitation, 196 mg ofcrystals precipitated, which were separated by decantation and dried.The specific rotation of these crystals was [α]_(D) ²⁵ =-24.6° (C=5, HCl1N), corresponding to an enantiomeric excess of 100% of (D) GLU in ayield of 17.6% with respect to the initial (D, L) GLU introduced.

EXAMPLES 32-35

In a manner similar to example 31, experiments were performed also atroom temperature, with 1 g, (D,L) GLU.H₂ O in 5 cc HCl 5N, in thepresence of seeds of (D,L) GLU.HCl and variable amounts of (L)ORN.HCl.The results are given in Table VI below.

                  TABLE VI                                                        ______________________________________                                        Resolution of (D,L) GLU HCl in the presence of (L)ORN.HCl                            wt (L)ORN.HCl                                                                            time     Precipitated crystals                              Example  (mg)         (days)   e.e. Yield                                     ______________________________________                                        32        50           2       87.4 21.15                                     33       200           3       98.8 17.1                                      34       200          14       92.8 19.5                                      35       500          10       98.4 20.4                                      ______________________________________                                    

EXAMPLE 36

1 g. (D,L) GLU.H₂ O and 25 mg (L) HIS.HCl were slurried in 5ml HCl 5Nand treated as in example 31. After 2 days 517 mg of a mixture of bigwell shaped bars and thin powdery plates had precipitated. These weredecanted and dried. The overall mixture was racemic. The twomorphological forms were then separated mechanically: the bars had aspecific rotation of [α]_(D) ²⁵ =-22.9 (c=5, HCl 1N) corresponding to anenantiomerical purity of 93.1%. (D) GLU, while the plates had [α]_(D) ²⁵=+24, corresponding to 97.6% pure (L) GLU.

EXAMPLES 37-42

In a manner similar to example 31 experiments were performed, also atroom temperature, with 1 or 1.5 g (D,L) GLU.H₂ O in 5 cc HCl 5N, in thepresence of seed crystals of (D,L) GLU.HCl and of different additives insolution. The results are given in Table VII below:

                  TABLE VII                                                       ______________________________________                                        Resolution of (D,L) GLU.HCl in the presence of various additives                   wt                   wt                                                  Ex-  (D,L)                addi-      precipitated                             am-  GLU.H.sub.2 O        tive time  crystals                                 ple  (g)       Additive   (g)  (days)                                                                              e. e.                                                                              yield                               ______________________________________                                        37   1         (L)HIS.HCl 0.5  14    96.1 21.8                                38   1         (L)HIS.HCl 0.8  14    99.2 20.3                                39   1.5       (L)HIS.HCl 0.8   6    94   24.5                                40   1         (L)CYS.HCl 0.8   2    99.2 16.9                                41   1         (L)THR     0.5  18    96.3  8.3                                42   1         (L)SER     0.2   4    58.5  3.3                                ______________________________________                                    

We claim:
 1. A process for resolution of a mixture of D- and L-forms ofasparagine, whereby the ratio of one desired enantiomorph to the otherundesired enantiomorph of said asparagine is increased in thecrystalline compound obtained, as compared to the ratio in the startingmaterial, which process comprises forming a supersaturated solution ofsaid mixture, adding D-glutamic acid, D-aspartic acid or D-glutamine ascrystallization inhibitor of the D-form when the L-form of asparagine isdesired, or including the L-form of these additives when the D-form ofasparagine is desired; and crystallizing a portion of the asparaginefrom said supersaturated solution.
 2. A process according to claim 1where, in addition, seeds of asparagine of the desired form are added tosaid supersaturated solution.
 3. A process for resolution of a mixtureof D- and L-glutamic acid hydrochloride, whereby the ratio of onedesired enantiomorph to the other undesired enantiomorph of saidglutamic acid hydrochloride is increased in the crystalline compoundobtained, as compared to the ratio in the starting material, whichprocess comprises forming a supersaturated solution of said mixture,adding D-Lysine HCl, (D)Lysine [(D)LYS], D-ornithine HCl, (D) ornithine[(D)ORN], (D) histidine HCl or (D) histidine [(D)HIS] as an inhibitor ofthe growing D-amino acid when the L-amino acid is desired, or a similaradditive in the L-form when the D-form is desired, and crystallizingpart of the compound from said supersaturated solution.
 4. A processaccording to claim 3, wherein also seed crystals of the desired form ofGLU.HCl are added during the crystallization step.
 5. A processaccording to claim 3, wherein both the D- form and the L- form areallowed to crystallize simultaneously, and where these are separatedaccording to the different morphology of the crystals.